PROJECT SUMMARY/ABSTRACT Recent advances in cancer immunotherapy have provided promising treatment options for patients with triple- negative breast cancer (TNBC). Despite overall success in treating these malignancies, immunotherapeutic ap- proaches face a number of unique challenges: (1) dose limitation due to off-target side effects, (2) additive toxicity of combination therapies, (3) and relatively low immunogenicity of breast cancer. To overcome these limitations, this proposal seeks to engineer probiotic strains of bacteria that selectively colonize breast cancer and locally release immunotherapeutics. The ultimate goal is to elicit more robust and diversified antitumor T cell immunity and promote the clearance of colonized primary and metastatic breast cancer lesions and systemically growing breast cancer-derived foci. The accompanying project will first focus on deciphering mechanisms that define the intratumoral tropism of the probiotic strain E. coli Nissle 1917 (EcN) by using antibody-mediated depletion ap- proaches and targeted genetic knockouts to pinpoint host immunological pathways that regulate tumor-specific growth. Using synthetic biology approaches, EcN will then be engineered to stably express and release check- point inhibitor nanobodies targeting CD47, PD-L1, and CTLA-4 locally inside of tumors. Pro-inflammatory cyto- kines will additionally be expressed to promote antigen presentation and enhance cytotoxic T cell responses. The primary innovations of this proposal are in the combined approach of both developing a better understanding of probiotic colonization of tumors, along with engineering probiotics as an immunotherapeutic delivery vector. Specifically, this approach has several advantages over current therapeutic strategies, including: (1) identifica- tion of novel EcN host strains and mechanistic understanding of their tumor colonization for further improvements in engineered therapies, (2) tumor-specific production of immunotherapeutics, (3) bacteria lysis that leads to effective release of novel immunotherapeutics and lipopolysaccharides (LPS) adjuvant, and (4) local delivery of novel immunotherapeutic combinations that are toxic to deliver systemically. This work seeks to overcome cur- rent limitations of immunotherapies, by providing a targeted vehicle to locally deliver immunotherapies that stim- ulate antitumor immunity while preventing systemic toxicity and mitigating immune-related adverse effects.